6-fluoro androstenes



fin wdsw s 7Q" fi-FLUORO AN DROSTENES' Raymond L. Pederson and Milton E.Herr, Kalamazoo, John C. Babcock, Portage Township, Kaiamazoo County,and J. Allan Campbell and John A. Hogg, Kalamazoo Township, KalamazooCounty, Mich, assignors to The Upjohn Company, Kalamazoo, Mich.,acorporation of Michigan No Drawing. Application June 9,1958

' Serial No. 740,530

' 38 Claims. c1. zen-497.3

thereof, 6-fluoro-1,4-androstadiene-3,17-diones, 6-fluoro1,4-androstadiene-3,1 1,17-triones, 6-fluo'ro-1 lfl-hydroxy-1,4-androstadiene-3,17-diones, 6-fluoroestradiols and the l7-acylatesthereof, 6-fiuoro-l1-ketoestradiols and the 17- acylates thereof,6-fluoro-1lfi-hydroxyestradiols and the l7-acylates thereof,6-fiuoroestrones, 6-fluoro-11-ketoestrones,6-fluoro-1lB-hydroxyestrones, and methods used in the preparationthereof. The novel steroids of this invention, the compounds of FormulaeII and HI, depicted and described in greater detail below, possessuseful therapeutic properties. The compounds of Formula II exhibitanabolic, antiestrogenic and androgenic activity. The compounds ofFormulae II and III possess antiosteoporotic, gonadotropini inhibiting,central nervous system regulating and blood lipid clearing properties.The compounds of Formulae II and III are useful in the treatment ofdebilitated, osteoporotic, hypogonadal, and atherosclerotic conditions.

Administration of the steroids of Formulae II and III can be inconventional dosage forms, such as pills, tablets, capsules, syrups orelixirs for oral use, or in liquid forms which are adaptable to thenatural and synthetic steroid hormones for injectable products.

The novel compounds of this invention are prepared according to thefollowing reaction scheme:

wherein R represents methyl or hydrogen, R represents hydroxy, OAcyl orketo and Zrepresents hydrogen, hydroxyl, or keto. The term Acyl as usedherein refers to an acyl radical of an organic carboxylic acid,preferably a hydrocarbon carboxylic acid, containing from one to twelvecarbon atoms, inclusive. I

In this application the wavy line (I) appearing at the 6-position is ageneric expression. including the alpha (a) and beta (6) configuration.

The starting steroids for the processes of the present invention, thecompounds of Formula I, e.-g., 6 -flu'oro- 17Bhydroxy-4-androsten-3-ones, 6-fluoro-17B-hydroxy- V 19 nor 4androsten-3-ones, 6-fluoro-17,8-hydroxy-4-androstene-3,11-diones,6-fluoro-l7fi-hydroxy-19-nor-4-androstene-3,1 l-diones,6-fluoro-11B,17B-dihydroxy 4-androsten-3-ones,6-fiuoro-1113,17B-dihydroxy-19-nor-4-androsten-3-ones, the *17-acylatesthereof, 6-fluoro-4-androstene- 3,17-diones,6-fiuoro-19-nor-4-androst'ene-3,17rdiones, 6-fluoro-4-androstene-3,11,17-triones, 6-fluoro-19-nor-4-androstene-3,11,17-triones, 6-fluoro-l 1,8-hydroXy-4-androstene-3,17-diones and6-fluoro-1lfi-hydroxy-19-nor-4-androstene-3,17-diones are disclosed inour copending application Serial No. 716,026, filed November 29, 1957.

One of the processes of ,the present invention comprises thefermentative dehydrogenation of the-com pounds of Formula I (where R ismethyl), for example,

6-fluoro-17B-hydroxy-4-androsten-3-ones and the 17-acyl-- ates thereof,6-fluoro-17,8-hydroXy-4-androstene-3,1l-diones and the 17-acylatesthereof, 6-fluoro-11,B,17,B-dihy droXy-4-and1'osten-3-ones and the17-acylates thereof, 6-

fluoro 4 androstene-3,17-diones, 6-fluoro-4-androstene- 3,11,17-trionesand 6-fluoro-11fi-hydroxy-4-androstene- 3,17-diones to obtain thecompounds of Formula II (where R is methyl), for example,6-fluoro-17B-hydroxy- 1,4-androstadien-3-ones,6-fluoro-17p-hydroXy-l,4-andro- 11B,17fi-dihydroXy-4-androsten-3-onesand the 17-acylatesthereof and6-fluoro-11fl-hydroxy-4-androstene-3,17-di-' ones, and Septomyxa isusedto efl'iect the dehydrogena tion, it is found to be advantageous touse with the subtrate and medium a steroid promoter, such as progester,

one, 3- \etobisnorr4-cholen-22-al, 3-ketobisnorcho1enic acid, 11fi,21-dihydroxy-1,4,1 7 (20)-pregnatrien-3-one, and the like.

When the starting steroid iscapable of possessing a 17-hydroxy or17-ester group, it is preferred touse a steroid possessing the17-hydroxyl group since dehydrogenation using microorganisms generallyefiects saponification of the 17-ester group. However, the 17-acylates,

. for example, the'17-acetate, the 17-propionate, and the17fi-dihydroxy-4-androsten-3-ones can be used as starting material. a

If desired, those compounds of Formula II (where R is methyl) whichcontain a 17-hydroxy group, such as 6 fiuoro 17Bhydroxy-1,4-androstadiene-3-ones, 6-

Patented Jan. 5,1959

alkaryl acid, e. g., benzoic, phenylacetic, B-phenylpropionic, om-, andp-toluic, a saturated dibasic acid (which can be converted intowater-soluble, e. g., sodium, salts), e. g., succinic, adipic; amonobasic unsaturated'acid, e. g., acylic, crotonic, undecylenic,propiolic, undecolic, cinnamic; dibasic unsaturated acids (which can beconverted into water-soluble, e. g., sodium, salts), e. g., maleic andcitraconic, or the acid anhydrides and acid halides thereof, can be usedto acylate the compounds of Formula II (where R is methyl) which containa l7-hydroxy group. If the esterifying agent is the free acid, thereaction is preferably effected in the presence of an esterificationcatalyst, for example, p-toluenesulfonyl chloride, trifluoroceticanhydride, p-toluene'sulfonic acid. trifiuoroacetic acid, sulfuric acid,and the like.

If the acylating' agent is an acid chloride or anhydride,

the reaction is preferably carried out in the presence of a tertiarybase, for example, pyridine, dimethylaniline, and the like.

If the corresponding acylating agent is solid, an inert solvent such astoluene, xylene or dioxane can be added to effect solution and toprovide a liquid esterification medium.

Alternatively the compounds of Formula I (where R is methyl) whichcontain a 17-hydroxyl or l7-acylate group, such as6-fluor0-17f3-hydroxy-4-androsten-3-ones and the 17-acylates thereof,6-fiu0ro-17B-hydroxy-4- androstene-3,l1-diones and the 17-acylatesthereof and 6-fiuoro-l1B,l7fi-dihydroxy-4-androsten-3-ones and the17-acylates thereof, can be subjected to chemical dehydro enation withselenium dioxide or selenious acid, according to procedures well knownin the art and furthermore illustrated in the examples below, to producethe compounds of Formula II (where R is methyl) which contain a17-hydroxyl or 17-acylate group, such as 6-fluoro-17B-hydroxy-1,4-androstadien-3-ones and the 17- acylates thereof,6-ii1ioro-l7B-hydroxy-l,4-androstadiene- 3,1l-diones and the l7-acylatesthereof, 6-fluoro-1lfl, l'lfl-dihydroxyd,4-andr'ostadien-3-ones and the17-acylates thereof.

Fe'imentative dehydrogenation of the compounds of Formula I (Where R ishydrogen), for example, 6-fiuoro- 17 8-hydroxy-19-nor-4-androsten-3-onesand the 17-acyl ates thereof, 6-fiuoro-17fl-hydroxy-l9-nor-4-androstene-3,ll-diones and the 17-acylates thereof, 6-fluoro-11,6,17;3-dihydroxy-19-nor 4-androsten-3-ones and the 17-acylates thereof,6-fiuoro-l9-nor-4-androstene-3,l7-diones, 6-fluore-'19-nor4-an'drostene-3,l1,17-triones and 6 fluoroF1lfl-hydroxy-19-nor-4-androstene-3,17-diones, is carried out in the samemanner'as described above for the fermentative dehydrogenation of thecompounds of Formula I (where R is methyl). The compounds of Formula II(where R is hydrogen) thus formed are unstable andspontaneously-rearrange to producethecompounds of Formula III, forexample 6-fluoro-estradiols, 6-fiuoroll-ketoestradiols,6-fluoro-1LB-hydroxyestradiols, 6-fluoroestrones,6-fluoro-11-ketoestrones and 6-fluoro-llfihydroxyestrones.

. When the compounds of Formula I (where R is hydrogen) contain anllfi-hydroxy group and Septomyxa is used to effect the dehydration, itis also found to be advantageous touse a steroid promoter, such as thosenamed above for the dehydrogenation of the compounds of Formula I(whereR is methyl).

If the 17-acylates of 6-fiuoro-17p-hydroxy-19mor-4- 7 hydrogen chloride.

7 4 Y androsten-3-ones,6-fluoro-17/3-hydroxy-19-nor-4-androstene-3,11-di0nes and 6-fluoro-1I3,17,B-dihydroxy-19-nor-4- androsten-3-ones are used as startingmaterials, saponification of the 17-ester group is generally effected.

The 17/3-acylates of the o-fluoroestradiols, 6-fluoro-11- ketoestradiolsand 6-fluoro-1lfl-hydroxyestradiols can then be obtained in thefollowing manner: The 6-fiuoroestradiols, 6-fluoro-ll-ketoestradiols ando-fluoro-llfl-hydroxyestradiols can be acylated, according to theprocedures described above for the acylation of 6-fiuoro-17fihydroxy-1,4-androstadien-3-ones, 6-fluoro-l7e-hydroxy-1,4androstadiene-3,1l-diones, and6-fiuoro-11B,17/3-dihydroxy-1,4-androstadien-3-ones, to obtain thecorresponding 3,17,8-diacylates of 6-fluoroestradiols, 6-fiuoro-11-ketoestradiols and 6-fiuoro-llp-hydroxyestardiols. The said3,17e-diacylates are then converted to the l7B-acylates of6-fluoroestradiols, 6-fluoro-11-ketoestradiols andllfl-hydroxyestradiols, by substituting the said 3,17B-diacylatesfor-the estradiol 3,17,8-diacylate disclosed in Example 2 of U. S.Patent 2,611,733 and following the procedure disclosed thereinselectively saponifying the acyl group at the 3-position. The preferredmethod for the preparation of the said 3,17/8-diacylates is to followthe procedure disclosed in Example 1 of U. S. Patent 2,611,733,substituting the 6-fluoroestradiols, 6-fluoro-11- ketoestradi-ols,6-fluoro-1lfl-hydroxyestradiols and the appropriate acid'halide for theestradiol and acid halide disclosed therein.

Chemical dehydrogenation of the compounds of Formula I (where R ishydrogen) which contain a 17-hydroxyl or 17-acylate group, such as6-fluoro-17fi-hydroxy- 19-nor-4-androsten-3-ones and the -17-acylatesthereof, 6-fluoro-17/3-hydroxy-19-nor-4 androstene 3,11 diones9-nor-4-androsten-3-onesand the 17-acylates thereof, to obtain thecompoundsof Formula III which contain a 17 -hydroxy or 17-acylate groupis carried outin the same manner described above for the conversion ofthe compounds of Formula I (where R is methyl) which contain al7-hydr0xy or 17-acylate group to the compounds of Formula II (where Ris methyl),which contain a 17- hydroxy or 17-acylate group. Here, as inthe fermentative dehydrogenation, the compounds ,of Formula II (where Ris hydrogen) which contain a 17'-hydroxy or 17-acylate group areunstable and spontaneously rearrange to produce the compounds of FormulaIII which contain a 17-hydroxy or 17-acylate group, such as6-fluoroestradiols and the l7-acylates thereof,-fluoro-lllietoestradiols and the 17-acylates' thereof, and 6-fluoro- 11fi-hydroxyestradiols and the l'Z-acylates thereof.

The foregoing compounds of Formulae I, II and III are all characterizedby the presence of a 6-fiuor0 substituent. It should be noted that theconfiguration of the fluorine at the 6-position can be either 6a or 65.Thus, substituting a 6/3-fluoro steroid as the starting material andfollowing the procedures hereinbefore described and as exemplifiedbelow, while maintaining near neutral reaction conditions, there isproduced as the final product of each example the correspondingo/i-epimer. Where the 6,8-epimer or a mixture predominating therein isemployed as the starting material, any subsequent reaction product canbe isolated either as the 6 8-epimer or the aforesaid mixture of 60cor6fi-epimers. A 6a-epimerized product can be obtained by treatment of the6,8-epimer (or a mixture of the 6:1- and 6,8-epimers) of the compoundsof Formula II at temperatures of zero degrees centigrade, or slightlyhigheror lower temperatures, in an organic solvent, such as chloroform,methylene chloride, ether, and the like, and in the presence of laprototropic agent (proton-donating agent) such as alcohols, organicacids, and the like, with a mineral acid, such as The mixture shouldpreferably be maintained at a temperature of zero degrees centigrade,although slightly higher or lower temperatures can be used, during theaddition of the acid. The reaction mixture can then be washed withsuccessive portions of dilute alkali and water, and then dried andevaporated under reduced pressure. The 6a-fluoro products can be recov--ered from the crude reaction product and purified by recrystallization.

Alternatively, the epimerization of the compounds of Formula II can beaccomplished with alkali. Bases, for example, solutions of sodiumhydroxide and potassium hydroxide, may be used to treat the 6 8 -epimerin solution in an organic solvent, such as methanol, to produce the6u-epimer. Y

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting. v

In the examples which follow, the Roman numeral following the name of acompound is used to indicate.

the relation of the compound to the reaction scheme depicted anddescribed above.

EXAMPLE 1 6a-flu0r0-11fi-hydroxy-I,4-andr0stadiene-3,17 dione (II)(biological dehydrogenation) Three lOO-milliliter portions of a medium,in 250- milliliter Erlenmeyer flasks, containing one percent glucose,two percent corn steep liquor (sixty percent solids) and tap water, areadjusted to a pH of 4.9. This medium is sterilized for 45 minutes atfifteen pounds per square inch pressure and inoculated with a one to twoday vegetative growth of Septomyxa afiinis A. T. C. C. 6737. TheErlenmeyer flasks are shaken at room temperature (iabout 26 to 28degrees centigrade) for a period of three ays. is used as an inoculumfor five liters of the same glucosecorn steep liquor medium which inaddition contains five milliliters of an *antifoam compound (a mixtureof lard oil and octadecanol). The fermentor is placed into thewater-bath, adjusted to 28 degrees centigrade and the contents stirred(300 R. P. M.) and aerated (0.3 liter air per minute to five liters ofbeer). After twenty hours of incubation, when a good growth has beendeveloped, one gram of 6u-finoro-llfl-hydroxy-4-androstene-3,l7- dione(I) plus one-half gram of 3-ketobisnor-4-cholen- 22-al dissolved insixteen milliliters of dimethylformamide is added and the incubationcarried out at the same temperature (28 degrees centigrade) and aerationfor a period of 48 hours (final pH 8.3). The mycelium is filtered offand extracted with three ZOO-milliliter portions of acetone. The beer isextracted with three one-liter portions of methylene chlorideandthereupon the aceton'e extracts and the extracts of the beer arecombined, dried over anhydrous sodium sulfate and evaporated and theresulting residue chromato'graphed over a Florisil anhydrous magnesiumsilicate column. The chromatographic column is eluted with Skellysolve Bhexanes plus increasmg proportions of acetone of from six to sixteenpercent. The fractions thus obtained are evaporated to dryness leavingresidues. The residues 'are subjected to infrared absorption analysisand those residues exhibiting the characteristic bands of6a-fluoro-11/3-hydroxy-1,4-androstadiene-3,l7-dion'e are combined andrecrystallized from methylene chloride-Skellysolve B hexanes to give6a-fluoro-1 LB-hydroxy-l ,4-androstadiene-3,17-dione (II), a crystallinesolid.

Instead of Septomyxa, species of other genera such as Corynebacterium,Didymella, Calonectria, Alternaria,

Colletotrichum, Cylindrocarpon, Ophiobolus, Fusarium,-

Listeria, Erysipelothrix, Mycobacterium, Tricothecium, Leptosphaeri'a,Cucurbitaria, Nocardia, and enzymes of fungi of the familyTuberculariaceae can be used to introduce a A -bond into6a-fluoro-1lp-hydroxy-4-androstene-3,l7-dione.

At the end of this period this 300-milliliter volume EXAMPLE 26a-flu0r'o-1,4-andr0stadiene-3,11,17 trione (l l (biologicaldehydrogenation) The Erlenmeyer flasks are shaken at room temperature(about 26 to 28 degrees centigrade) for a period of three days. Atthe'end of this period this 300-milliliter volume is used as an inoculumfor five liters of the same glucose-corn steep liquor medium which inaddition contains five milliliters of an antifoam compound (a mixture oflard oil and octadecanol). The fermentor is placed intothe water-bath,adjusted to 28 degrees centigrade and the contents stirred (300 R. P.M.) and aer-.

ated (0.3 liter air per minute to five liters of beer). After twentyhours of incubation, when a good growth has been developed, one gram of6a-fluoro-4-androstene- 3,11,17-trione (I) in sixteen milliliters ofdimethylformamide is added and the incubation carried out at the sametemperature (28 degrees centigrade) and aeration;

for a period of 48 hours (final pH 8.3). The mycelium' is filtered offand extracted with three ZOO-milliliter portions of acetone. The beer isextracted with three oneliter portions of methylene chloride andthereupon the acetone extracts and the extracts of the beer arecombined, dried over'anhydrous sodium sulfate and evaporated and theresulting residue chromatographed over a Florisil anhydrous magnesiumsilicate column. The chromatographic column is eluted with Skellysolve Bhexanes plus increasing proportions of acetone of from six to sixteenpercent. evaporated to dryness leaving residues. The residues aresubjected to infrared absorption analysis and those residues exhibitingthe characteristic bands of 6a-fluoro-1,4- androstadiene-B,11,17-trioneare combined and recrystallized from methylene chloride Skellysolve Bhexanes to give 6a-fiuoro-1,4-androstadiene-3,11,17-trione (11),

EXAMPLE 3 6 a-fl uoro-I -hydroxy-1,4-andr0stadiene-3,1l-dione' (II)(biological dehydrogenation) Three IOO-milliliterportions of a medium,in 250-1 milliliter Erlenmeyer flasks, containing one percent glu-.cose, two percent corn steepliquor (sixty percent solids) and tap water,are adjusted to a pH of 4.9. This medium is sterilized for 45 minutes atfifteen pounds per square inch pressure and inoculated with a one to twoday vegetative growth of'Septomyxa afiinis A. T. C. C. 6737. TheErlenmeyer flasks are shaken at room tem-' perature (about 26 to 28degrees centigrade) for a period of three days.

the same glucose-corn steep liquor medium which in addition containsfive milliliters of an antifoam compound (a mixture of lard oil andoctadecanol). The fermentor is placed into the water-bath, adjusted to28 degrees; centigrade and the contents stirred (300 R. P. M.) andaerated (0.3 liter air per minute to five liters of beer).

After twenty hours of incubation, when a good growth;

has been developed, one gram of 6a-fluoro-l78-hydroXy-4-androstene-3,ll-dione (I) in sixteen .r'nilliliters ofdimethylformamide is added and the incubation. carried out at the sametemperature (28 degrees centigrade) and aeration for a period of 48hours (final pH'8.3).-I The mycelium is filtered off 1 and extractedwiththree;

The fractionsthus obtained are At the end of this period this 300-milliliter volume is used as an inoculum for five liters ofZOO-milliliter portions of acetone. The beer is extracted with threeone-liter'portions of methylene chloride and thereupon the acetoneextracts and the extracts of the beer are combined, dried over anhydroussodium sulfate and evaporated and the resulting residue chromatographedover a Florisil anhydrous magnesium silicate column. The chromatographiccolumn is eluted with Skellysolve B hexanes plus increasing proportionsof acetone of from six to sixteen percent. The fractions thus obtainedare evaporated to dryness leaving residues. The residues are subjectedto infrared absorption analysis and those residues exhibiting thecharacteristic bands of 6a fiuoro-173-hydroxy-1,4-androstadiene-3,1l-dione are combined and recrystallizedfrom methylene chloride- Skellysolve B hexanes to give 6a-fiuoro17/3-hydroxy-1,4 androstadiene-3,11-dione (II), a crystalline solid.

Instead of Septornyxa, species of other genera such as those disclosedin Example 1 can be used to introduce a A -bond intoGot-fluoro-17,3-hydroxy-4-androstene-3,l1- dione.

' Instead of 6c:-fluoro-17,8-hydroxy-4 androstene-3,11- dione, the 17-estcrs thereof can be usedsuch as the 17- a'cetate, the" 17-propionate,the 17-butyrate, the 17-isobutyrate, and the like. However, in thesecases the ester group is generally saponified during the fermentationprocess.

EXAMPLE 3A 0.85 gram of '6ot-fiuoro-17/3-hydroxy-1,4-androstadiene-3,11-dione is dissolved in three milliliters of pyridine. This solutionis then treated with 1.5 milliliters of propionic anhydride and allowedto stand for five hours. The reaction mixture is cooled and ninemilliliters of water is added dropwise with stirring, causing theprecipitation of a solid. After one hour the mixture is filtered and theprecipitate dried in vacuo at seventy degrees centigrade to give6a-fluoro-17fl-hydroxy-1,4-androstadiene-3,11-dione 17-propionate (II),a crystalline solid.

Similarly, by allowing6a-fiuoro-l7fi-hydroxy-1,4-androstadiene-3,11-dione to react With theappropriate hydrocarbon carboxylic acids, or the acid anhydrides andacid halides thereof, there are produced other 17-acylates of 6ot-fiuoro17,8 hydroxy 1,4 androstadiene 3,11- dione such asGas-fluoro-17fi-hydroxy-1,4-androstadiene- 3,11-dione 17-formate,6a-fiuoro-17fl hydroxy 1,4-androstadiene-3,1l-dione 17-acetate, 6afiuoro 175-hydroxy-1,4 androstadiene 3,11 dione 17 butyrate,6ozfluoro-17B-hydroxy-L4 androstadiene 3,11 dione 17- valerate,6rx-tluoro-l7l8 hydroxy 1,4 androstadiene- 3,11-dione 17-hexanoate,6a.-fluoro-17fl hydroxy 1,4- androstadien'e-3,11-di0ne 17 laurate, 60cfluoro 17phydroxy 1,4- androstadiene 3,11 dione 17 trimethylacet'ate,6a-fluoro-l7B-hydroxy-1,4 androstadiene 3,11- dione l7-iso'butyrate,Gar-fluoro-17,8-hydroxy-1,4-androstadiene-3,1'1-dione 17-is'ovalerate,6a-fiuoro-17fl-hydroxy- 1,4-androstadi'ene-3,lI-dione l7-tertiarybutylacetate, 6afluoro-17flhydroxy 1,4 androstadiene 3,11 dione17-(,d-cyclopentylpropionate), -6a-fluoro 1718 hydroxyl,4-androstadiene-3,11 dione 17 -cyclohexane carboxylate,6a-fiuoro-17fl-hydroxy 1,4-- androstadiene 3,11- dionel7-cyclohexylacetate, 6a fluoro 17o hydroxy- 1,4-androstadiene 3,11dione 17 benzoate, 6a-fiuoro- 17 6 hydroxy 1,4 7 androstadiene 3,11dione l7- phenylacetate, 60 --fiuoro 173- hydroxy 1,4 androstadiene-3,1-1-dione 1-7-(fi-phenylpropionate), 6a fluoro- 1-7fl-hydr0 xy 1,4androstadiene 3,11 dione 17-(o-, m-,'p toluate'), 6a-fluoro 175 -hydroxy1,4 androstadiene ll l-dione '17-hemisuccinate, 6u-fluoro-17p-hydroxy-1,4 androstadiene-lll-dione 17.hemiadipate, 6oz-fluo'ro-l7a-hydroxy 1,4androstadiene 3,11 dione 17'-acrylate, 6a-fluoro-17/8+hydroxy -.1,4androstadiene 3,11-dione 17-croton'ate, 60c -.fluoro 175 hydroxy-1,4-androstadiene-3J1-dione 17' undecylenate, 6a ,fluorofluoro-17fl-hydroxy-L4 androstadiene 3,11 dione 17- citraconate.

EXAMPLE 4 I 6 a-fluoro-J 7,8-hydn0xy-1,4-andr0stadien-3-one (II(chemical dehydrogenation) A mixture of 0.918 gram (0.003 mole) of6a-fluoro- 1713-hydroxy-4-androsten-3.-one (1) dissolved in eighteenmilliliters of tertiary butyl alcohol and 0.18 milliliter of acetic acidWas heated together with 021 gram of purified selenious acid at refluxunder stirring for fivehours. Thereafter 0.12 gram of purified seleniousacid was added and the mixture heated under reflux, with stirring, for afurther period of sixteen hours. The mixture was then cooled, filteredand the filtrate concentrated to dryness in vacuo. The residue Wasdissolved in fifty milliliters of methylene chloride, washed with Water,twice with saturated sodium bicarbonate solution, again with water,driedfover sodium sulfate and evaporated to dryness in vacuo. dissolvedin sixty milliliters of benzene and chromatographed on fifty grams ofFlorisil (synthetic magnesium silicate). The chromatographic column waseluted as follows:

Fraction 1 (1000 milliliters) four percent acetone Fraction 2 1000milliliters)- Skellysolve B hexanes plus six percent acetone SkellysolveB hexanes plus Fractions 3-43 (SO-milliliter fractions)-Skellysolve Bhexanes plus eight percent acetone The residues from fractions 13through 37 were identified by infrared absorption and melting point andwere combined to give 0.361 gram of product which was recrystallizedfrom methylene chloride-Skellysolve B hexanes to yield 0.308 gram of6tx-fiuoro-l7fi-hydroxy-l,4- androstadiene-3-one (II), melting at182-184 degrees centigrade.

Analysis.-Calcd. for C H FO C, 74.97; H, 8.08. Found: C, 74.79; H, 8.31.

Substituting 6rx-fiuoro-17fl-hydroxy 4 androsten 3- one 17-acylates forGOL-flUOIO-17,8-hYd1'OXY-4-and1'0sllel1- 3-one is productive of thecorresponding 6oc-fil1010-l7flhydroxy-l,4-androstadien-3-one17-acylates, for example, the 17-acetate, the 17-propionate, the17-hemisuc-. cinate, the 17-benzoate, and the like.

EXAMPLE 5' 6 oc-flllOHO-I 1,8,1 7 3-dihydlfo icy-l ,4-andr0stadien-3-one (11) (chemical dehydrogenation) V A mixture of 100 milligrams of6a-flUOI0-llfi,l7fl dihydroxy-4-androsten-3-one (I) dissolved insix,.milliliters of tertiary butyl alcohol and 0.55 milliliter of aceticacid is heated together with thirty milligrams of selenium dioxide toapproximately degrees centigrade' under stirring for a period of about24 hours. Thereafter another thirtynnilligram portion of seleniumdioxide is added and the mixtureheated under continuous stirring for afurther period of about 24 hours. The mixture is thencooled, filteredand evaporated. The residue is dissolved in fifty milliliters ofmethylene chloride, Washed with sodiumbicarbonate solution, Water, driedover sodium sulfate and the solvent is evaporated therefrom. The residueis chromatographed over a Florisil anhydrous magnesium silicate column.The chromatographic column is eluted with Skellysolve B hexanes plusincreasing proportions of acetone of from five to twenty percent. Thefractions thus obtained are evaporated to dryness leavingresidues. Theresidues are subjected to infrared absorption analysis and those Theresidue was 9 7 residues exhibiting the characteristic bands of6a-fluoro 115,17fi-dihydroxy 1,4 androstadien 3 oneare combined andrecrystallized from methylene chloride-Skellysolve. B hexanes to give6a-fluoro-11B,17fl-dihydroxy- 1,4-androstadien-3-one (II), a crystallinesolid.

Substituting 6a-fluoro-l15,175-dihydroxy 4 androsten-3-one 17-acylatesfor 6a-fluoro-11fi,17 8-dihydroxy- 4-androsten-3-one is productive ofthe corresponding 6afluoro-l1 8,17/3-dihydroxy-1,4-androstadien 3 one17- acylates, for example, the 17-acetate, the 17-propionate, thel7-hemisuccinate, the 17-benzoate, and the like.

ExAMPLE'6 6a-fluoroestrone (III) (biological dehydrogenation) Three100-milliliter portions of a medium, in 250- milliliter Erlenmeyerflasks, containing one percent glucose, two percent corn steep liquor(sixty percent solids) and tap water, are adjusted to a pH of 4,9. Thismedium is sterilized for 45 minutes at fifteen pounds per square inchpressureand inoculated with a one to; two day vegetative growth ofSeptomyxa affinis A. T. C. C. 6737. The Erlenmeyer flasks are shaken atroom temperature (about 26 to 28 degrees centrigrade) for a period ofthree days. At the end of this period this 300-milliliter volume is usedas an inoculum for five liters of the same glucosecorn steep liquormedium which in addition contains five milliliters of an antifoamcompound (a mixture of lard oil and octadecanol). The fermentor isplaced into the water-bath, adjusted to 28 degrees centrigrade and thecontents stirred (300 R. P. M.) and aerated (0.3 liter air per minute tofive liters of beer). After twenty hours of incubation, when a goodgrowth has been developed, one gram of6u-fluoro-l9-nor-4-androstene-3,17-dione (I) in sixteen milliliters ofdimethylformamide is added and the incubation carried out at the sametemperature (28 degrees centrigrade) and aeration for a period of 48hours (final pH 8.3). The mycelium is filtered off and extracted withthree ZOO-milliliter portions of acetone. The beer is extracted withthree one-liter portions of methylene chloride and thereupon the acetoneextracts and the extracts of the beer are combined, dried over anhydroussodium sulfate and evaporated and the resulting residue chromatographedover a Florisil anhydrous magnesium silicate column. The chromatographiccolumn is eluted with Skellysolve B hexanes plus increasing proportionsof acetone of from five to twenty percent.

The fractions thus obtained are evaporated to dryness leaving residues.The residues are subjected to infrared absorption analysis and thoseresidues exhibiting the characteristic bands of a-fluoroestrone arecombined and recrystallized from acetone-hexane to give6ot-fiuoroestrone (III), a crystalline solid. I

Instead of Septomyxa, species of other genera such as those disclosed inExample 1 can be used to convert 6afluoro-19-nor-4-androstene-3,17-dioneto 6a-fluoroestrone.

EXAMPLE 7 6m-fluor0-11-ket0estrone (Ill (biological dehydrogenation)Three IOU-milliliter portions of a medium in 250- milliliter Erlenmeyerflasks, containing one percent glucose, two percent corn steep liquor(sixty percent solids) and tap water, are adjusted to a pH of 4.9. Thismedium is sterilized for 45 minutes at fifteen pounds per square inchpressure and inoculated with a one to two day vegetative growth ofSeptomyxa affinis A. T. C. C. 6737. The Erlenmeyer flasks are shaken atroom temperature (about 26 to 28 degrees centrigrade) for a period ofthree days. At the end of this period this 300-milliliter volume is usedas an inoculum for five liters of the same glucose-corn steep liquormedium which in addition contains five milliliters of an.antifoamcompound (a mixture of lard oil and octadecanol). The fermentor isplaced into the water-bath, adjusted to 28 degrees centigrade and theketoestrone.

: stirring for a period of about 24 hours.

contents stirred (300 R. P. M.) and aerated (0.3 liter air per minute tofive liters of beer). After twenty hours of incubation, when a goodgrowth has been developed one gram of6a-fiuoro-19-nor-4-androstene-3,11,17-trione in sixteen milliliters ofdimethylformamide is added and the incubation carried out at the sametemperature (28 de-' grees centigrade) and aeration for a period of 48hours (final pH 8.3). The mycelium is filtered off and extracted withthree ZOO-milliliter portions of acetone. The beer is extracted withthree one-liter portions of methylene chloride and thereupon the acetoneextracts and the extracts of the beer are combined, dried over anhydroussodium sulfateand evaporated and the resulting residue chromatographedover a Florisil anhydrous magnesium silicate column. The chromatographiccolumn is eluted with Skellysolve B hexanes plus increasing proportionsof acetone of from five to twenty percent. The fractions thus obtainedare evaporated to dryness leaving residues. The residues are subjectedto infrared absorption analysis and those residues exhibiting thecharacteristic bands of 6a-fluoro-11-ketoestrone are combined andrecrystallized from acetone-hexane to give 6a-fluoro-11-ketoestrone(III), a crystalline solid.

Instead of Septomyxa, species of other genera such as those disclosed inExample 1 can be used to convert6ozfluoro-19-nor-4-androstene-3,11,17-trione to 6a-fluoro-1 1- EXAMPLE 86a-fluoroestradiol (HI) (chemical dehydrogenation) A mixture of 100milligrams of 6a-fluoro-17B-hydroxy- 19-nor-4-androsten-3-one (I)dissolved in six milliliters of tertiary butyl alcohol and 0.55milliliter of acetic acid is heated together with thirty milligrams ofselenium dioxide to approximately 75 degrees, centigrade underThereafter another thirty-milligram portion of selenium dioxide is addedand the mixture heated under continuous stirring for a further period ofabout 24 hours. The mixture is then cooled, filtered and evaporated todryness. The

residue is dissolved in 75 milliliters of methylene chloride, washedwith sodium bicarbonate solution, water, dried over sodium sulfate andthe solvent is evaporated therefrom. The thus-obtained residue ischromatographed over a Florisil anhydrous magnesium silicate column. Thechromatographic column is eluted with Skellysolve B hexanes plusincreasing proportions of acetone of from five to twenty percent. Thefractions thus obtained are evaporated to dryness leaving residues. Theresidues are subjected to infrared absorption analysis and thoseresidues exhibiting the characteristic bands of 6a-fluoroestradiol arecombined and recrystallized from methylene chloride-Skellysolve Bhexanes to give 60cfluoroestradiol (III), a crystalline solid.

Substituting 6ot-fluoro-17fi-hydroxy-19 nor 4 andro sten-3-one17-acylates for ,6u-fluoro-l7 8-hydroxy-l9-nor- 4-androsten-3-one isproductive of the corresponding 60cfluoroestradiol l7fl-acylates, forexample, the 17B-acetate, the l7B-propionate, the 17,8-hemisuccinate,the 17,B-b enzoate, and the like.

- EXAMPLE 9 6 a-fluoro-Z 1 B-hydroxyestradiol (III) (chemicaldehydrogenation) A mixture .of milligrams of6oc-fil1Q1O-11]3,17)9-dihydroxy-19-nor-4-androsten-3-one (1) dissolvedin six milliliters of tertiary butyl, alcohol and 0.55 milliliter of'The five to twenty percent.

.1. A. washed, with sodium bicarbonate solution, water, dried over;sodium sulfate and .the solvent is evaporated therefrom. Thethus-obtainedv residue is chromatographed.

over a Florisil anhydrous magnesium silicate column. The chromatographiccolumn is eluted with Skellysolve B hexanes, plus increasing proportionsof. acetone of from The fractions thus obtained are evaporated todryness leaving residues. The residues are subjected to infraredabsorption analysis and those residues exhibiting the characteristicbands of 6oL-flllGl'O- ll-hydroxyestradiol are combined andrecrystallized from methylene chloride-Skellysolve .B. hexanes to giveGa-fluoro-lhydroxyestradiol (III), a crystalline solid.

Substitutingfid fluoro 115,175 dihydroxy 19-nor.-

droxy-l9rnor-4-androsten-3-one is productive of the correspondin6a-fiuoro-115-hydroxyestradiol 175-acylates,

, for example, the 175-acetate, the 175-propionate, the

steroid end-product, such-as, for example, 65-fluoro-1,4-

androstadiene-3,11,17-trione (Example 2), 65-fluoro-175-hydroxy-1,4-androstadiene-3,1l-dione (Example 3), 65- fluoro 175 hydroxy1,4 androstadiene 3,11 dione 17-propionate (and other 17-acylatesthereof) (Example 3A), 65 fluoro 175 hydroxy 1,4 androstadiene 3- one(and the 17-acylates thereof) (Example 4), 65-lluoro- 115,175 dihydroxy1,4 androstadien 3 one (and the 17-acylates thereof) (Example 5),65-fluoroestrone (Example 6), 65-fiuoro-ll-ketoestrone (Example 7), 65-fluo roestradiol (and the 175-acylates thereof) (Example 8) and65-fiuoro-llfi-hydroxyestradiol (and the 175- acylates thereof).(Example 9).-

' EXAMPLE 11 Isomarization of the 65-flu0r0 steroids to thecorresponding 6a-flu0r0 steroids Illustratively, this reaction iscarried out as follows:

A solution of one gram of 65-fluoro-115-hydroxy-1,4-

androstadiene-3,17-dione in 100 milliliters of chloroform and 0.1milliliter of alcohol is cooled to approximately minus ten degrees in anice-salt bath and a stream of anhydrous hydrogen chloride is greatlybubbled through the solution for about 2.5 hours Whilst maintaining thetemperature between approximately minus five and minus fifteen degreescentigrade. The solution is then washed with dilute sodium bicarbonatesolution and water, dried over anhydrous sodium sulfate, and evaporatedunder reduced pressure. Crystallization of the residue fromacetone-Skellysolve B hexanes yields6a-fluoro-115-hydroxy-1,4:androstene-3,17-dione, of Example 1.

In a similar manner other 65-fluoro steroids, for example,65-fluoro-1,4-androstadiene 3,11,17 trione, 65- fiuoro 175 hydroxy 1,4androstadiene 3,11 dione (and the 17-acylates thereof),65-fluoro-175-hydroxy- 1,4-androstadien-3-one (and the'17-acylatesthereof), and 65 fiuoro 115,175 dihydroxy 1,4 androstadiene-3- one (andthe 17-acylates thereof) can be converted to their correspondingmat-analogues.

This application is a continuation-in-part of application Serial No;716,026, filed February 19, 1958, which is in turn acontinuation-in-part of application Serial No; 699,505, filed-November29, 1957, now abandoned.

It is to be understood that the'invention is not to be limited. to theexact detailsof operationlor; exactvcompounds shown and;,described,, asobvious modifications. and equivalents will be, apparent to: oneskilled; 11131116,; art, and theinvention is? therefore to be limitedonly'by;

the scope of the appended claims.

We claim: 1.,A 6-fluoro-1-dehydro compound of the following, formula:

wherein R is selected from the group consisting ofhydroxyl, OAcyl, andketo, and term Acyl representingthe acyl radical of a hydrocarboncarboxylic acid containing;-

from one to twelve carbon atoms, inclusive, and Z is selected from thegroup consisting of hydrogen, hydroxyl, and keto. 7

2. 6-fluoro-175-hydroxy-1,4-androstadien-3-one.

3. 6u-fluoro l-hydroxy-1,4-androstadien-3-one.

. 6-fluoro-1,4-androstadiene-3,17-dione.6a-fluoro-1,4-androstadiene-3,17-dione.6-fiuoro-175-hydroxy-1,4-androstadiene-3,1l-dione.

ooogoxm-ts 6-fluoro-1,4-androstadiene-3,11,17-trione. 6xfluoro-.1,4-androstadiene-3,l1,17-trione. '10. 6-fiuoro-1 15-hydroxy-1,4-androstadiene+3,17-dionei 11. 6a fluorohydroxy-l,4-androstadiene- 3,1 7- dione.

13. 6ix-fluoro 115,175- dihydroxy-1,4-androstadien-3 one.

atoms, inclusive.

1S. 6m-fluoro-175-hydroxy-1,4-androstadiene-3-one 17 propionate.

l6. 6-fluoro- 115,175-dihydroxy-1,4-androstadiene13} one l7-acylate inwhich the acyllradical is that ofa hydro? carbon carboxylic acidcontaining from one toitwelve carbon atoms, inclusive. e

17., 6a-fluoro 115,175 dihydroxy-l,4-androstadien-3- one 17-propionate.

1S. 6-fiuoro-l75-hydroxy-1,4-androstadiene3,1 l dione 17-acylate inwhichthe acylradical-is that ofahydrocarbon carboxylic acid containingfrom one to; twelve carbon atoms, inclusive. V

19. 6a-fiuoro-175-hydroxy-1,4-androstadiene6,l l-dione 17propionate-.- 1

20. A 6-fluoro compound of'the'following fomula:

F wherein R is'selected from the groupconsistingiof liy droxyl, OAcyl,and keto, the term Acyl representing the: acyl radical of a hydrocarboncarboxylic acid containing from one to twelve carbon atoms,inclusive,.and: Z isse- 6a-fluoro-175-hydroxy 1,4-androstadiene-3,1ldione. l

12. 6-fiuoro-1 15,175-dihydroxy-1,4-androstadien-3 -on e.

14. 6-fiuoro-175-hydroxy-1,4-androstadien-3-one--17-ae-- ylate in,whi'chthe acyl radical is that of-ahydrocarbom carboxylic acidcontaining from one to twelve carbon:

14 lected from the group consisting of hydrogen, hydroxyl, 33.fi-fluorestradiol 17,53-acy1ate in which the acyl radand keto. ical isthat of a hydrocarbon carboxylic acid containing 21. 6-fluoroestradio1.from one to twelve carbon atoms, inclusive. 22. Ga-fluoroestradiol. 34.Goa-fluoroestradiol 17fi-propionate. 23. 6-fluoro-1l-ketoestradiol. 535. 6-fluoro-11-ketoestradiol 17fi-acylate in which the 24.6a-fiu0ro-1l-ketoestradiol. acyl radical is that of a hydrocarboncarboxylic acid con- 25. 6-fluoro-1lp-hydroxyestradiol. taining from oneto twelve carbon atoms, inclusive. 26. 6a-fluoro-1lfi-hydroxyestradiol.36. 6a-fluoro-1l-ketoestradiol 17/3-propionate. 27. 6-fiuoroestrone. 37.6-fluoro-1lfi-hydroxyestradiol 17B-acylate in which 28.6a-fluoroestrone. 10 the acyl radical is that of a hydrocarboncarboxylic acid 29. 6-fluoro-11-ketoestrone. containing from one totwelve carbon atoms, inclusive. 30. 6u-fll1010-11-k6t085i10118. 38.6a-fluoro-1lfl-hydroxyestradiol 17,8-propionate.

31. 6-fluoro-1lfi-hydroxyestrone. 32. 6a-fiuoro-1lfi-hydroxyestrone. Noreferences cited.

UNITED STATES PATENT OFFICE Certificate of Correction Patent No.2,867,630 January 6, 1959 Raymond L. Pederson et al.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring correction and that the saidLetters Patent should read as corrected below.

Column 3, line 23, for trifluorocetic read trifluoroacetic-; column 4,lines 34 and 35, for that portion of the formula reading-dihydroxy-9-nor4- read--dihydroxy-19-nor-4--; column 7, line 72, forthat portion of the formula reading -17ahydroxyread -17,8-hydroxy-Signed and sealed this 12th day of May 1959.

Attest: KARL H. AXLINE, ROBERT C. WATSON,

Attesting Oficer. Uommz'ssz'oner of Patents.

1. A 6-FLUORO-1 COMPOUND OF THE FOLLOWING FORMULA:
 26. A 6-FLUOROCOMPOUND OF THE FOLLOWING FORMULA: